International Journal of Pharmaceutics 577 (2020) 119070

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Colon-targeting of progesterone using hybrid polymeric microspheres T

improves its bioavailability and in vivo biological efficacy
⁎
Hytham H. Gadallaa, Fergany A. Mohammeda, Ahmed M. El-Sayeda, Ghareb M. Solimana,b,
a
Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
b
Department of Pharmaceutics, Faculty of Pharmacy, University of Tabuk, Tabuk, Saudi Arabia

A R T I C LE I N FO A B S T R A C T

Keywords: This study aims to enhance progesterone (PG) oral bioavailability via its incorporation into hybrid colon-tar-
Site-specific delivery geted pectin/NaCMC microspheres (MS) cross-linked with Zn2+ and Al3+. The MS were characterized for
Pectin particle morphology, encapsulation efficiency, swelling behavior, drug release, mucoadhesivity and colon-spe-
Sodium carboxymethylcellulose cific degradability. Response-surface methodology was adopted to optimize the fabrication conditions.
Mucoadhesion
Enhancement of in vivo drug performance was evaluated through pharmacokinetic and pharmacodynamic stu-
Endometrial proliferation
Colon targeting
dies. The optimized formulation was typically spherical with a mean diameter of 1031 µm and drug entrapment
efficiency of 88.8%. This formulation exhibited pH-dependent swelling, negligible drug release in simulated
Chemical compounds studied in this article: gastric fluid and sustained-release pattern in simulated small intestinal fluid with a mean t50% of 26.5 h. It also
Progesterone (PubChem CID: 5994) showed prolonged and preferential adhesion to rat colonic mucosa, as well as expedited degradation in presence
Pectin (PubChem CID: 441476) of rat caecal contents. The MS significantly increased the area under the curve and mean residence time by 1.8
Sodium carboxymethylcellulose (PubChem and 2.3-fold, respectively compared to the free drug. Orally administered MS showed ~10 times increase in
CID: 23706213) myometrial thickness compared with the drug suspension and elicited uterine responses very similar to that
Zinc acetate dihydrate (PubChem CID: obtained parenterally. These results confirm the ability of this new carrier system to improve the oral bioa-
2724192) vailability of PG and attain adequate clinical efficacy.
Aluminum sulfate (PubChem CID: 24850)
Aluminum chloride (PubChem CID: 6451240)
Tween 80 (PubChem CID: 5284448)

1. Introduction Sriamornsak et al., 2006). These carriers are inert, biocompatible,

biodegradable and have minimal cost (Mennini et al., 2008; Sharma
Site-specific drug delivery is one of the most promising strategies to et al., 2013). In this context, pectin (Pc) and sodium carbox-
enhance drug efficacy and reduce off-target side effects through ymethylcellulose (NaCMC) are specifically attractive due to their re-
achieving drug release at the diseased tissues (Alange et al., 2017; Sinha sistance to hydrolysis in the upper GIT and specific hydrolysis in the
and Kumria, 2003). The colon represents an attractive site for specific colon by colonic microflora (Darvari and Hasirci, 1996; Sinha and
drug delivery thanks to its longer transit time and reduced proteolytic Kumria, 2003). Moreover, the slow, pH-dependent swelling properties
activity as compared to the small intestine (Maestrelli et al., 2008; of NaCMC can help delay the drug release until reaching the colon
Sinha and Kumria, 2003; Wang et al., 2016). Colon targeting is also (Agarwal et al., 2015). Yet, rapid Pc dissolution in the upper GI fluids is
useful to attain optimal effects of drugs requiring delayed absorption for a challenging hurdle that must be addressed before it can be applied in
therapeutic considerations, such as nocturnal asthma or anginal colon-targeted vehicles (Andishmand et al., 2017). To circumvent this
therapies (Kaur et al., 2014; Sinha and Kumria, 2003). Further, the problem, different approaches including cross-linking of pectin chains
prolonged colonic transit time might improve the oral bioavailability of and/or combination with other polymers were reported (Das et al.,
poorly water-soluble drugs by increasing the time available for their 2011; El-Gibaly, 2002; Gadalla et al., 2016b; Paharia et al., 2007;
dissolution and absorption (Wang et al., 2016; Wang et al., 2015). Prezotti et al., 2014; Rubinstein et al., 1993). Amidation of pectin
Polysaccharide-based microspheres have the potential to further chains was also reported to improve its properties and make it more
prolong colonic residence time with a massive increase in surface area tolerant to different pH and ionic strengths encountered within the GIT
exposed to bacterial digestion (Jose et al., 2010; Rodrı́guez et al., 1998; (Kowalonek, 2017).

⁎
Corresponding author at: Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt.
E-mail address: [email protected] (G.M. Soliman).

https://doi.org/10.1016/j.ijpharm.2020.119070
Received 16 November 2019; Received in revised form 13 January 2020; Accepted 18 January 2020
Available online 23 January 2020
0378-5173/ © 2020 Elsevier B.V. All rights reserved.
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Progesterone (PG) is a natural female sex hormone extensively Table 1

employed as a hormone replacement therapy in a wide array of gyne- 23 factorial design independent parameters, studied levels and composition of
cological disorders, as well as a standard care measure to support the the optimized formulation (M7).
luteal phase in assisted reproduction (Ruan and Mueck, 2014). Un- Factors Low level (−1) Middle level (0) High level (+1) Optimum Level
fortunately, it suffers very short elimination half-life (~19–95 min) and
low oral bioavailability, presumably due to its limited aqueous solubi- X1 1.5 1.75 2.0 1.5
X2 0.05 0.06 0.07 0.07
lity as well as extensive intestinal and hepatic metabolism. This greatly
X3 0.025 0.088 0.15 0.15
limits its clinical utility as an oral medication (Beltsos et al., 2014;
Dollery, 1999; Vinarov et al., 2018). The primary forms of PG in clinical X1: Polymer concentration (% w/v) (Pc/NaCMC, 1:1).
practice now are oily IM injection and intravaginal inserts, with com- X2: Zn(CH3COO)2 concentration (M).
parable efficacy (Beltsos et al., 2014; Zarutskie and Phillips, 2009). X3: Al2(SO4)3 concentration (M).
Both routes suffer inconvenience issues, which highlights the need for
an effective oral alternative. PG is a BCS class II drug, which implies 2.2. Microsphere preparation
that drug dissolution is the major step determining its rate and extent of
absorption (Amidon et al., 1995). Targeting of PG to the colon is as- Hybrid Pc/NaCMC MS loaded with PG were prepared by the mod-
sumed to prolong its GI residence time, avert intestinal deactivation and ified ionotropic gelation method previously reported by El-Gibaly
ultimately enhance its oral bioavailability. Although limited fluid vo- (2002) using two cross-linking agents. An appropriate amount of PG
lume and increased viscosity of colonic contents might adversely affect (1%, w/v) was dispersed in 0.3% w/v aqueous Tween® 80 solution in
drug dissolution, we believe that the benefits of targeting PG to the deionized (DI) water under magnetic stirring until a uniform dispersion
colon outweigh the drawbacks, given its very low plasma minimum was formed. Then, Pc/NaCMC blend (1:1 ratio) was added to the drug
effective concentration (2–5 ng/ml). Further, colon targeting could dispersion to form PG-loaded hydrogel. The homogenous, bubble-free
avoid intestinal PG metabolism and enhance drug dissolution through hydrogel was added in a drop-wise manner at an average rate of 1 ml/
the much longer colon transit time (Amidon et al., 2015; Jameela et al., min into 25 ml of the gently-stirred cross-linking solution [Zn
1998). (CH3COO)2 and Al2(SO4)3 in DI water]. The addition was made using a
The literature shows very limited number of studies attempting to disposable syringe attached to 1 mm-inner diameter tip with the falling
enhance PG oral bioavailability. So far, most researchers focused on distance fixed at 5 cm. The instantaneously-formed MS were allowed to
increasing PG aqueous solubility via different solubilization techniques cure in the cross-linking solution for 10 h and then washed three times
without examining the actual enhancement of drug performance in vivo with DI water and dried in air for 48 h. Empty MS made in the same
(Kim et al., 2010; Nandi et al., 2003; Schwarz et al., 2017; Vinarov method but with no drug served as a blank. Eight batches were pre-
et al., 2018; Wiedmann et al., 2002). In our previous work, we de- pared by varying the experimental conditions according to 23 factorial
monstrated that bi-polymeric Pc/NaCMC microspheres can serve as a design; each batch was prepared in triplicate (Table 1 and Supple-
promising platform to improve the oral bioavailability of hydrophobic mentary, Table A).
drugs via enhancement of their colonic permeability (Gadalla et al.,
2016a). However, these formulations were unable to sufficiently pre- 2.3. Particle size and shape
serve the drug load in the upper GI conditions, which reduced the re-
sidual drug amount reaching the colon. The size of MS from different batches was measured from images
The main focus of this study was to optimize the drug-retaining captured by Leica EC3 camera connected to a Leica optical microscope
ability of our previously-reported system to better endure the upper GI (Leica Microsystems, Heerbrugg, Switzerland). The MS were dried at
conditions and maximize drug concentration reaching the colon. room temperature for 48 h at room temperature to constant weight.
Moreover, several studies previously reported lack of correlation be- After complete drying, fifty MS were randomly picked from each batch
tween in vitro release data and actual in vivo performance, presumably and photographed. Morphological analyses were performed on the di-
due to many enzymatic and biological variables which are overlooked gitalized images using image analyzing software (ImageJ, version
in the in vitro experiments (Mittal et al., 2007; Sandor et al., 2002). 1.48). The shape of MS was described in terms of aspect ratio (AR) and
Therefore, the potential of the optimized formulation to improve PG circularity (C).
oral absorption and the effects on myometrial thickness were assessed
through pharmacokinetic and pharmacodynamic experiments.
2.4. Scanning electron microscopy (SEM) studies

2. Materials and methods Samples of completely dry MS were mounted onto stubs and sputter
coated with gold in a vacuum evaporator. The surface topography of
2.1. Materials MS was examined and photographed under scanning electron micro-
scope (Jeol, JSM-5200, Tokyo, Japan) at 15 keV.
Micronized PG was a gift from Pharco Pharmaceuticals Inc.
(Alexandria, Egypt). Genu pectin (type LM-104 AS) with 36% degree of 2.5. Entrapment efficiency and drug loading capacity
esterification (DE) and 14% degree of amidation (DA) was purchased
from Copenhagen pectin (Copenhagen, Denmark). Low-viscosity grade An exact amount of MS (25 mg) was finely powdered, suspended in
NaCMC was purchased from Sigma Chemical Co. (St. Louis, Mo). 50 ml of absolute ethanol and stirred for 2 h to ensure complete drug
Injectable estradiol benzoate of commercial grade (Folone®) was ob- dissolution. Samples were then filtered through 0.45 µm filter discs.
tained from Misr Pharmaceutical Industrial Co. (Cairo, Egypt). Zinc After proper dilution, the filtrate was assayed spectrophotometrically at
acetate dihydrate, aluminum sulfate, sodium chloride, monobasic so- 242 nm. Blank MS treated under the same conditions were taken as a
dium phosphate, disodium hydrogen phosphate, hydrochloric acid, control. The entrapment efficiency (EE) and drug loading capacity (DL)
Tween® 80 and absolute ethanol were obtained from Prolabo Chemicals were calculated from the following equations:
Co. (Cairo, Egypt). All chemicals used were of analytical grade and used
Actual drug content
as received. Entrapment Efficiency (EE %) = × 100
Theoritical drug content
(1)

2
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Actual drug mass 2.9. In vitro drug release in simulated colonic environment
Drug Loading capacity (DL %) = × 100
Total microsphere mass
(2) Colon-specific release of PG from the optimized MS formulation was
studied in presence of rat caecal contents as a source of pectinolytic
enzymes (Paharia et al., 2007). In brief, five male Wistar rats weighing
200–220 g with no prior drug treatment were maintained on regular
2.6. In vitro drug release studies
diet. The pectinolytic enzymes were induced by 1-ml daily oral ad-
ministration of 2% aqueous pectin solution for 7 consecutive days.
The in vitro drug release was investigated using the USP XXV dis-
solution apparatus (Erweka, DT-D6, Heusenstamm, Germany) with the Immediately prior to the experiment, rats were sacrificed and the ab-
domen was opened. The caecum was legated, cut and immediately
dissolution basket assembly at a rotational speed of 50 rpm and tem-
perature of 37 ± 0.2 °C. Three dissolution media with pH 1.2, 5.5 and transferred into PBS bubbled with CO2 to provide anaerobic conditions.
The caecal bag was opened and the contents were weighed, homo-
7.4 were used sequentially to account for the stomach, duodenum,
lower small intestine and colon, respectively (Crison, 1999). An accu- genized and suspended in phosphate buffer (60 mM, pH 7) to give a
final concentration of 1%, w/v caecal contents. The suspension was
rately weighed amount of MS equivalent to 10 mg PG was placed in the
dissolution baskets and immersed in 500 ml of simulated gastric fluid then filtered, sonicated for 10 min in ice bath to disrupt the bacterial
cells and centrifuged at 6000 rpm for 10 min. The supernatant was then
(SGF, 0.1 M HCl containing 0.2% NaCl, pH 1.2) for 2 h. Subsequently,
MS were immediately transferred into 500 ml of fresh simulated small used as a simulated colonic fluid (SCF). For the initial 2.5 h, the drug
release experiment was conducted as described above, after which the
intestinal fluid (SSIF, 60 mM phosphate buffer, pH 5.5). After 0.5 h, MS
were finally transferred into 500 ml of fresh SSIF (60 mM phosphate SCF was added until the end of study. CO2 was continuously supplied
into the medium to maintain anaerobic environment similar to that of
buffer, pH 7.4) for the rest of study. All release media contained 0.02%
the colon. At different time points, 5-ml samples were withdrawn and
Tween® 80 to ensure sink condition. At specified time intervals, 5-ml
replaced with an equal volume of the corresponding medium. Samples
samples were withdrawn with an equal volume of fresh medium being
were centrifuged at 6000 rpm for 10 min, filtered through 0.45 µm
replenished to maintain constant volume of the release medium. Sam-
filter discs and assayed for their PG content spectrophotometrically at
ples were assayed spectrophotometrically for PG content at 242 nm. All
242 nm against a similarly-treated blank.
dissolution experiments were performed in triplicate.

2.10. In vivo pharmacokinetic study

2.7. Equilibrium swelling studies
The pharmacokinetic parameters of the optimized MS formulation
In order to assess the MS ability to withstand the different pH were determined through a randomized parallel study using healthy
conditions encountered along the GIT, their swelling behavior was male New Zealand rabbits weighing 1.8–2 kg. Rabbits were randomly
studied in enzyme-free SGF (pH 1.2) and enzyme-free SSIF (pH 7.4). divided into two groups, each of 3 rabbits. After 3 days of acclimati-
Constant weight of blank MS (100 mg) was placed in the baskets of the zation, rabbits were fasted for 12 h prior to drug administration but had
USP XXV dissolution apparatus and immersed in 500 ml of each free access to water. Food was not allowed for further 12 h following
medium maintained at 37 ± 0.2° C. At different time intervals, MS drug administration. A dose of 10 mg/kg body weight of free micro-
were removed, dried and their weight changes were recorded. The nized PG or an equivalent dose of the optimized formulation (corre-
equilibrium swelling index of each formula was then calculated as sponding to 200 mg human dose) were filled into hard gelatin capsules
following: of size 4 and administered to rabbits by the aid of a stomach tube. Blood
samples of 1 ml were withdrawn from the eye vein into heparinized
Wt − W0
Swelling Index (SI) = centrifuge tubes at the following time points: pre-dose, 1, 2, 3, 4, 6, 8,
W0 (3) 10, 12, 18, 24 and 30 h following drug administration. Blood samples
were centrifuged at 5000 rpm for 15 min and plasma was separated.
where W0 is the initial weight of the dry MS and Wt is the weight of MS
Liquid–liquid extraction with diethyl ether was performed twice to
after swelling for time t.
recover PG from plasma before analysis. The ether phase was then se-
parated, evaporated and the residues were reconstituted in 500 µl of
2.8. Ex vivo mucoadhesion mobile phase. Samples were then analyzed by high performance liquid
chromatography (HPLC, Shimadzu 2010A, Kyoto, Japan) using a RP-
The mucoadhesive properties of the optimized MS formulation were C18 column (Phenomenex, 150 × 4.6 mm, 5 µm). The mobile phase
evaluated under different GI conditions using the wash-off method consisted of filtered, degassed mixture of acetonitrile and water (70:30,
previously reported by Agarwal et al. (2015) with minor changes. All v/v) pumped at a flow rate of 1 ml/min. The column was maintained at
animal experiments were performed in accordance with the animal room temperature and the effluent was delivered directly into the UV-
ethical guidelines approved by Assiut University, Egypt. The experi- diode array detector (model K-2500, Knauer, Berlin, Germany). The
ment was conducted in enzyme-free SGF (pH 1.2) and enzyme-free SCF detection wavelength was set at 240 nm. The lower limit of detection
(pH 7.0) for tissues obtained from stomach and colon of male Wistar for this method was found to be 1 ng/ml. Drug recovery from plasma
rats, respectively. Briefly, rats were sacrificed and tissues from different samples was > 92%. Plasma drug concentration versus time profiles
parts of their GIT were excised and immediately transferred into ice- were then plotted and analyzed using the non-compartmental approach
cold phosphate-buffered saline (PBS). Tissues were carefully opened, with oral input.
evacuated and washed several times with cold PBS. Segments of equal
size were fixed on glass slides with the mucosal surface kept upward. 2.11. In vivo progestational activity
Afterwards, 25 mg of MS were hydrated in each corresponding medium
and allowed to adhere to the mucosal surface by applying a weight of The in vivo performance of the optimized formulation was further
10 g for 5 min. The slides were placed horizontally in the vessels of USP assessed through the well-established endometrial proliferation assay
XXV dissolution apparatus and MS were forced to wash-off under a commonly used for screening and comparing progestational activity
stirring speed of 50 rpm in 500 ml of each corresponding medium kept (McPhail, 1934). Briefly, immature New Zealand female rabbits
at 37° C. The time required for complete detachment of MS from mu- weighing 700–800 g were given a once-daily IM injection of 5 µg es-
cosal surfaces was then recorded. tradiol benzoate in 0.5 ml of sesame oil for 6 consecutive days.

3
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Thereafter, the estrogen-primed rabbits were randomly divided into MS were relatively larger in size with a uniform spherical shape and
five groups (n = 3). Group I received no further drug treatment and rough surface (Fig. 1B), which are believed to be due to the insoluble
served as a negative control. Group II, III and IV received an oral dose of nature of PG embedded in the MS matrix. Favorably, the optimized MS
5 mg PG in the form of aqueous suspension, oily solution in sesame oil, exhibited no surface folds, cracks or fissures, which anticipated slower
and the optimized MS formulation, respectively. The fifth group (V) swelling and drug release.
received an equal PG dose in sesame oil intramuscularly (positive
control). This treatment was continued for another 5 days. Twenty-four
3.3. Entrapment efficiency and drug loading
hours after the last PG dose, rabbits were sacrificed and both uterine
horns were removed and fixed in 10% neutral buffered formalin. Tis-
The drug EE for the different batches was high and ranged from 80.1
sues from the middle uterine segment were then dehydrated in graded
to 97.8% w/w, mainly due to the low aqueous solubility of PG in ad-
alcohol series, cleared with methyl benzoate, embedded in paraffin
dition to the rapid formation of drug-sealing cross-linked network
wax, sectioned at 4 μm and stained with Hematoxylin and Eosin as well
(Supplementary, Table A). High drug loading capacity is a desirable
as Periodic Acid Schiff (PAS) stains for further histological examination.
feature to minimize the burden of excipients. The optimized formula-
Endometrial proliferation scores from 0 (no response) to 4 (maximum
tion exhibited adequate drug loading capacity of 35.5%, confirming its
proliferation) were then assigned for each animal and averaged for each
potential for drug delivery applications.
group according to the modified McPhail scale. The myometrial
Analyzing the factors affecting MS drug entrapment, we found that
thickness was also calculated from the digitalized images obtained by
the EE decreased significantly upon increasing Al3+ ion concentration
optical microscopy (Leica Microsystems, Heerbrugg, Switzerland).
(Supplementary, Fig. A1). As an acidic salt, Al2(SO4)3 concentration
controls both the solution pH and the cross-linking degree. More acidic
2.12. Statistical analysis
cross-linking solutions promote contraction of polymeric chains and
subsequently inhibit matrix swelling resulting in a limited void volume
Statistical and graphical analyses of the factorial design results were
for drug entrapment (Alange et al., 2017). Additionally, the expulsion
performed using the software package Minitab (Minitab software, ver-
of water during gelation caused a convective loss of the drug and fur-
sion 17.1). For statistical comparisons, data was analyzed by one-way
ther decreased the EE. This comes in accordance with the relatively
analysis of variance (ANOVA) with a post-hoc test using GraphPad Prism
smaller size of MS prepared at the higher Al3+ level.
(GraphPad Software, version 7.0) and differences were considered
significant at p < 0.05. All data are presented as mean ± standard
deviation (SD) of at least 3 independent replicates. 3.4. In vitro drug release

3. Results and discussion Despite the highly variable pH values and transit times reported in
literature for different GI segments, we tried to select the conditions
3.1. Preparation of PG-loaded MS that are in consensus with most studies. Accordingly, the drug release
was tested in 3 dissolution media of pH 1.2, 5.5 and 7.4 in sequential
Among the different techniques employed for MS preparation, ionic manner. The cumulative release profile of the optimized MS shows a
gelation is advantageous for its safety, simplicity and relatively high sigmoidal release pattern, indicating pH-dependency. The faster drug
product yield (Cerciello et al., 2017). Herein, the MS yield was never release in SSIF relative to that observed in SGF could be explained in
less than 85% in any batch, which emphasizes the cost-effectiveness of virtue of the acid-resistant nature of the matrix polymers, as well as
this preparation process. It is also noteworthy that this method is not their enhanced swellability in near neutral pH (Seeli et al., 2016).
involving any deleterious organic solvents or harsh conditions that may In SGF, the optimized formulations showed minimal drug release
compromise the formulation safety. (~5%) compared to 59% for the free drug after 2 h. However, abrupt
MS formation was mediated by ionic cross-linking of the negatively drug release could be noticed at pH 5.5, possibly due to greater matrix
charged polysaccharide chains with the counter-ions (Bera et al., 2017). swelling and increased drug solubility at higher pH. The saturation
Diffusion of Zn2+ and Al3+ cations to the carboxyl residues of Pc or solubilities of PG in SGF and SSIF were measured to be 20.3 ± 2.3 and
NaCMC yielded an insoluble matrix that effectively entrapped the drug 124.8 ± 0.3 µg/ml, respectively. The optimized MS released less than
and sustained its release (El-Gibaly, 2002). Cross-linking of polymer 15% of its drug load after 2.5 h, which implied that it can successfully
chains as well as the compatibility of PG with matrix ingredients were prevent premature drug release in the upper GI fluids (Fig. 2). The drug
assured through FT-IR and DSC analyses in our previous work (Gadalla continued to be released until a sustained (plateau) phase was evident
et al., 2016a). The factorial multivariate analysis was employed to after 4 h. The drug amount released initially is believed to be due to
optimize the fabrication conditions (Table 1 and Supplementary, Table dissolution of surface or near surface drug; however, the drug release
A) and hence, a formulation of desired characteristics was achieved from the MS core was much slower and predominates afterwards.
(Table 1). Having in mind that it takes 3–4 h for a dosage form to reach the colon
(Rubinstein, 2005) and considering the minimal drug release observed
3.2. Morphological evaluation throughout this time interval, we assumed that these MS are capable of
holding most of their drug load until being released specifically in the
The average diameter of optimized MS (M7) was 1031 ± 19 µm. colon upon matrix degradation.
These MS exhibited a significantly smaller size than all other batches, The polymer concentration played the most crucial role affecting
presumably due to the combined effect of using the lower polymer the drug release rate. Increasing the polymer concentration within the
concentration and the greater cross-linking density achieved with the study limits markedly hastened the drug release and resulted in shorter
higher level of both cross-linkers (Alange et al., 2017). As gelation t50% (Supplementary, Table A and Fig. A2). Higher polymer con-
proceeds, more water is expelled from the MS matrix due to greater centration promotes matrix swelling due to the extremely hydrophilic
number of cross-links formed, resulting in smaller particles. Further, all nature of Pc and NaCMC, which open-up the matrix structure and cause
formulations exhibited good sphericity as indicated by the corre- rapid drug release (Dafe et al., 2017). Moreover, t50% increased sensibly
sponding values of aspect ratio (AR) and circularity (C), which were upon increasing Zn2+ ion concentration. This effect was more promi-
close to unity (Supplementary, Table A). nent at the lower polymer concentration, possibly due to achieving the
Under SEM, blank MS appeared small, distorted in shape with a proper polymer/cross-linker ratio, which allowed more polymeric
collapsed center and smooth surface (Fig. 1A). In contrast, PG-loaded chains to be cross-linked and further reduced drug release.

4
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Fig. 1. SEM images of (A) blank MS and (B) optimized PG-loaded MS (M7) at different magnification powers (1) × 35, (2) × 75 and (3) × 1000.

Fig. 3. Swelling behavior of the optimized MS formulation in enzyme-free SGF

(pH 1.2) and enzyme-free SSIF (pH 7.4).
Fig. 2. Cumulative drug release profiles of free micronized PG and the opti-
mized MS formulation (Drug release tested sequentially in enzyme-free SGF (pH
1.2, 2 h), enzyme-free SSIF (pH 5.5, 0.5 h) and enzyme-free SSIF (pH 7.4) for
3.6. Ex vivo mucoadhesion
the rest of study).
At pH 7, the vast majority of MS remained attached to the colonic
mucosa for up to 30 h until they completely disintegrated to form a gel-
3.5. Equilibrium swelling studies
like adhesive layer. In contrast, the MS were entirely washed off from
the stomach mucosa at pH 1.2 after 2–2.5 h. The much longer adhesion
Swelling behavior of the optimized MS was assessed in SGF (pH 1.2)
time noticed with the colonic mucosal tissue could be explained by the
and SSIF (pH 7.4) at 37 °C. Interestingly, it exhibited pH-responsive
higher degree of carboxyl group ionization at neutral pH which allows
swelling; an attractive add-on feature that further enhances site-speci-
for greater matrix swelling and hence, stronger entanglement with
ficity of this formulation. The MS swelled minimally in SGF (Fig. 3) and
mucin chains (Bigucci et al., 2008; Gadalla et al., 2016b). Conversely,
remained intact with no signs of erosion for up to 24 h, reflecting their
the negative charge of the polymeric chains tends to be shielded in
stability in acidic media (Agarwal et al., 2015; Gadalla et al., 2016a).
acidic pH, which reduces their ability to interact with mucin. This pH-
However, when placed in SSIF, the MS showed an initial swelling lag
dependent mucoadhesion is a valuable feature enabling preferential
for 1.5 h (Fig. 3). Once the matrix front was opened, water started to get
residence of MS at the colon with subsequent release of the loaded drug.
into the MS extensively with exponential rise in SI until equilibrium was
A good correlation was found between the swelling and mucoadhesive
established, after which MS started to erode and disintegrate slowly. MS
properties of this matrix, suggesting that polymer swelling is the most
swelling occurs due to deprotonation of the polymeric carboxylate
influential parameter affecting MS mucoadhesion.
groups in neutral or alkaline pH, which increases the repulsion among
polymer chains, loosens the MS structure and allows more water to
diffuse into the matrix (Andishmand et al., 2017; Bajpai et al., 2006). 3.7. In vitro drug release in simulated colonic environment
Increased Al3+ levels contribute to lower SI and more prolonged
equilibrium time due to greater cross-linking density (Supplementary, Caecum represents a diverse environment with a wide variety of
Table A). The correlation between MS swelling and drug release cor- anaerobic bacteria that produce enzymes involved in the metabolism of
roborates our hypothesis that matrix swelling is a crucial parameter various carbohydrates (Singh et al., 2015). Among different animal
that must be properly controlled in designing effective colon-targeted models, rats show the greatest similarity to human cecal microflora and
vehicles. share the same most common bacterial species (Ravi et al., 2008).

5
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Table 2
Pharmacokinetic parameters of PG after oral administration of free micronized
PG and the optimized MS formulation to rabbits (n = 3).
Pharmacokinetic parameters Free PG powder PG-loaded MS

Cmax (µg/ml) 1.61 ± 0.04 1.37 ± 0.02***

Tmax (h) 6 ± 0 12 ± 0***
Ka (h−1) 0.30 ± 0.08 0.17 ± 0.01**
t½a (h) 2.4 ± 0.6 4.0 ± 0.1**
MRT (h) 8.3 ± 0.11 19.4 ± 0.01***
AUC (µg·h/ml) 19.4 ± 0.9 34.9 ± 0.3***
FR (%) – 180

** p < 0.01.
*** p < 0.001.

was achieved 6 h post-dose. Afterwards, it rapidly declined to un-

detectable levels after 24–30 h. The short biological half-life of PG is
Fig. 4. In vitro drug release profiles from the optimized MS formulation in responsible for this rapid plasma disappearance following its oral ad-
enzyme-free media or in presence of 1% w/v rat caecal contents (Drug release ministration. In contrast, the plasma drug concentration increased tar-
tested sequentially in enzyme-free SGF (pH 1.2, 2 h), enzyme-free SSIF (pH 5.5,
dily after administration of PG-loaded MS owing to slower drug release
0.5 h) and SCF (pH 7.0, with or without 1% w/v rat caecal contents) for the rest
and absorption. The plasma drug level gradually increased to attain a
of study).
Cmax of 1.37 µg/mL after 12 h, from which it slowly decreased but was
still detectable at high concentration of 0.5 µg/ml after 30 h.
Initially, the release curves in SGF were identical, with less than 7% Pharmacokinetic analysis of the obtained data revealed that the
drug released from MS (Fig. 4). However, when SCF containing rat mean residence time (MRT) of PG from the MS formulation was sig-
caecal contents was added, the drug release was significantly enhanced nificantly increased by 2.3 folds, showing the MS ability to markedly
as compared to that observed in the enzyme-free medium (p < 0.05). extend PG effect. This could be attributed to the slow drug release from
This trend was consistent until the end of study, at which 61% of PG the MS matrix, which acts as a drug reservoir controlling the release
was released in SCF as compared to only 47% in enzyme-free medium. rate and allowing for gradual drug absorption. Moreover, a PG relative
The higher drug release in SCF could be attributed to the abundant bioavailability of 180% was observed for the MS. This enhanced bioa-
microflora present in caecum, which promote enzymatic digestion of vailability might be due to the strong mucoadhesive properties as well
pectin (Alange et al., 2017; Paharia et al., 2007). This enhanced drug as colon-specific drug release which extended the GI residence time and
release in simulated colon environment confirms the colon-specific increased the time available for drug dissolution and absorption
degradability of the matrix. It was previously reported that the percent (Gadalla et al., 2016b; Nappinnai et al., 2013).
drug released in simulated colonic environment is a function of rat cecal
contents in the release medium (Jain et al., 2007; Prasad et al., 1998).
Typically, the human colon has much greater enzyme concentration 3.9. In vivo progestational activity
due to higher polysaccharide consumption rate, suggesting that com-
plete PG release could be achieved when these microspheres are ad- Motivated by the promising results of the pharmacokinetic study,
ministered to human subjects (Prasad et al., 1998). we proceeded to test whether this could reflect an improved therapeutic
response in vivo. This study was also extended to include more chal-
lenging treatment groups in order to assess the actual performance of
3.8. In vivo pharmacokinetic study
the MS formulation relative to the currently available forms in the
market.
The mean plasma concentration-time profiles after oral adminis-
Histological examination and scoring of female rabbit uteri using
tration of the optimized MS formulation and micronized PG powder are
McPhail scale provide a useful mean for evaluating the progestational
depicted in Fig. 5, whereas the derived pharmacokinetic parameters are
activity of progestins in a semi-quantitative manner (DeManno et al.,
listed in Table 2. Following oral administration of free PG, the plasma
2003). Estrogen-priming is reported to mimic the natural in vivo hor-
drug concentration increased rapidly until a peak level of 1.61 µg/ml
monal release pattern, increase the uterus size and enhance uterine
response to PG treatment (McPhail, 1934). This biological assay relies
on the pronounced transformation and development of the different
uterine layers in response to PG action. Typically, exposure of pre-
mature female rabbits to PG induces hyperplasia of the endo- and
myometrial epithelial cells, increased neovascularization, hyperaemia
of blood vessels in addition to endometrial stromal proliferation (Beri
et al., 1998). Moreover, glandular development starts to take place,
indicated by increase in the number and/or size of the endometrial
uterine glands (Abd Elkareem, 2017).
Microscopic examination of uterine tissue specimens revealed that
rabbits in group I (negative control) showed no endometrial prolifera-
tion with a compact endothelium, continuous epithelial lining and
minimal myometrial thickness, thus assigned a McPhail score 0 (Fig. 6A
and Table 3). The uteri of rabbits treated with PG oral suspension
(group II) showed slight proliferation (crypt formation) and incon-
Fig. 5. Mean plasma drug concentration-time profile after oral administration siderable myometrial thickening. In contrast, the group receiving oral
of free micronized PG and the optimized MS formulation to healthy male rab- PG in oily vehicle (group III) showed distinct endometrial response with
bits (n = 3). moderate glandular and microvascular development as well as a

6
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Fig. 6. Photomicrographs of uterine cross-sections from estrogen-primed immature rabbits (n = 3 per group) receiving: (I) No treatment (negative control), (II) PG
oral suspension, (III) PG oily solution, (IV) PG-loaded optimized MS and (V) IM PG injection (positive control). (A) H & E stain: Left panel × 25 (scale bar = 1 mm),
right panel × 100 (scale bar = 200 µm) and (B) PAS stain × 200 (scale bar = 100 µm).

Table 3
Evaluation of progestational activity in estrogen-primed immature female rabbits following administration of 5 mg PG in different forms.
Treatment (n = 3) Myometrial thickness (µm) Folds increase of myometrial thickness1 Endometrial response (McPhail index)

Estrogen only (Negative control) 293 ± 42 – 0

Oral suspension 310 ± 48 1 1.2 ± 0.4
Oral oily solution 448 ± 51** 9.1 2.8 ± 0.4
Oral MS formulation (M7) 462 ± 33*** 9.9 3.5 ± 0.5
IM (Positive control) 467 ± 49*** 10.2 4.0 ± 0

Folds increase of thickness for each group = (thickness of this group − thickness of negative control)/(thickness of oral suspension group − thickness of negative
control).
** p < 0.01.
*** p < 0.001 compared to the oral suspension group.
1
Relative to the group receiving oral suspension.

7
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

significantly thickened myometrium. A full and maximal proliferative doi.org/10.1016/j.ijpharm.2020.119070.

effect was noticed in group V following IM administration of PG
(McPhail score 4). Herein, the glands became of lower abundance but References
penetrated deeply into the stroma, thus reaching the myometrium with
further myometrial thickening. The microvascular development was Abd Elkareem, M., 2017. Morphological, histological and immunohistochemical study of
evidenced by the formation of large microvessels and subepithelial the rabbit uterus during pseudopregnancy. J. Cytol. Histol. 8, 443.
Agarwal, T., Narayana, S.G.H., Pal, K., Pramanik, K., Giri, S., Banerjee, I., 2015. Calcium
capillary plexuses. Likewise, rabbits receiving the optimized MS for- alginate-carboxymethyl cellulose beads for colon-targeted drug delivery. Int. J. Biol.
mulation (group IV) elicited nearly the same response. Using PAS stain, Macromol. 75, 409–417.
the uterine glands of group IV could be seen more clearly as PAS ne- Alange, V.V., Birajdar, R.P., Kulkarni, R.V., 2017. Functionally modified polyacrylamide-
graft-gum karaya pH-sensitive spray dried microspheres for colon targeting of an
gative while the endothelial basal lamina appeared as PAS positive anti-cancer drug. Int. J. Biol. Macromol. 102, 829–839.
(Fig. 6B). Compared to the oral suspension group, the optimized MS Amidon, G.L., Lennernas, H., Shah, V.P., Crison, J.R., 1995. A theoretical basis for a
showed ~10 times more increase in myometrial thickness from baseline biopharmaceutic drug classification-the correlation of in-vitro drug product dissolu-
tion and in-vivo bioavailability. Pharm. Res. 12, 413–420.
owing to the enhanced drug dissolution and absorption (Table 3). These Amidon, S., Brown, J.E., Dave, V.S., 2015. Colon-targeted oral drug delivery systems:
results confirm that oral PG delivery with an efficacy comparable to design trends and approaches. Aaps Pharmscitech 16, 731–741.
that obtained intramuscularly can be achieved with the developed oral Andishmand, H., Tabibiazar, M., Mohammadifar, M.A., Hamishehkar, H., 2017. Pectin-
zinc-chitosan-polyethylene glycol colloidal nano-suspension as a food grade carrier
multi-particulate system.
for colon targeted delivery of resveratrol. Int. J. Biol. Macromol. 97, 16–22.
Bajpai, S.K., Saxena, S.K., Sharma, S., 2006. Swelling behavior of barium ions-crosslinked
4. Conclusion bipolymeric sodium alginate–carboxymethyl guar gum blend beads. React. Funct.
Polym. 66, 659–666.
Beltsos, A.N., Sanchez, M.D., Doody, K.J., Bush, M.R., Domar, A.D., Collins, M.G., 2014.
Mucoadhesive microspheres were prepared and optimized for Patients’ administration preferences: progesterone vaginal insert (Endometrin®)
achieving colon-specific release of PG with the ultimate goal of en- compared to intramuscular progesterone for Luteal phase support. Reprod. Health
hancing its oral bioavailability and maximizing therapeutic outcomes. 11, 78.
Bera, H., Nadimpalli, J., Kumar, S., Vengala, P., 2017. Kondogogu gum-Zn+2-pectinate
Dual-targeting strategies were employed to ameliorate MS colon-tar- emulgel matrices reinforced with mesoporous silica for intragastric furbiprofen de-
geting capability. High and consistent product yield was achievable livery. Int. J. Biol. Macromol. 104, 1229–1237.
with the simple and mild ionic gelation procedure. The influence of Beri, R., Kumar, N., Savage, T., Benalcazar, L., Sundaram, K., 1998. Estrogenic and pro-
gestational activity of 7α-methyl-19-nortestosterone, a synthetic androgen. J. Steroid
some formulation parameters on the microsphere drug entrapment and Biochem. Mol. Biol. 67, 275–283.
release characteristics was investigated to optimize fabrication condi- Bigucci, F., Luppi, B., Cerchiara, T., Sorrenti, M., Bettinetti, G., Rodriguez, L., Zecchi, V.,
tions. The optimized formulation exhibited excellent features, such as 2008. Chitosan/pectin polyelectrolyte complexes: Selection of suitable preparative
conditions for colon-specific delivery of vancomycin. Eur. J. Pharm. Sci. 35, 435–441.
high drug loading, uniform spherical shape, pH-responsive swelling, Cerciello, A., Del Gaudio, P., Granata, V., Sala, M., Aquino, R.P., Russo, P., 2017.
sustained drug release, site-specific mucoadhesion and specific de- Synergistic effect of divalent cations in improving technological properties of cross-
gradation in the colon. Moreover, it significantly prolonged the drug linked alginate beads. Int. J. Biol. Macromol. 101, 100–106.
Crison, John R., 1999. Developing dissolution tests for modified release dosage forms:
residence time and enhanced its absorption in vivo, with a relative
general considerations. Dissol. Technol. 6 (1), 5–9. http://www.dissolutiontech.com/
bioavailability of 180% compared to the free micronized drug. After DTresour/299articles/299Crison.htm http://www.dissolutiontech.com/DTresour/
oral administration to rabbits, the optimized MS elicited uterine re- 299articles/299Crison.htmhttps://doi.org/10.14227/DT210314P110.14227/
sponses very similar to that obtained intramuscularly. Altogether, this DT060199P5.
Dafe, A., Etemadi, H., Dilmaghani, A., Mahdavinia, G.R., 2017. Investigation of pectin/
polymeric matrix represents an attractive carrier for oral delivery of PG starch hydrogel as a carrier for oral delivery of probiotic bacteria. Int. J. Biol.
and offers a therapeutic alternative to the less acceptable parenteral Macromol. 97, 536–543.
route. Darvari, R., Hasirci, V., 1996. Pesticide and model drug release from carbox-
ymethylceullose microspheres. J. Microencapsul. 13, 9–24.
Das, S., Chaudhury, A., Ng, K.-Y., 2011. Polyethyleneimine-modified pectin beads for
CRediT authorship contribution statement colon-specific drug delivery: In vitro and in vivo implications. J. Microencapsul. 28,
268–279.
DeManno, D., Elger, W., Garg, R., Lee, R., Schneider, B., Hess-Stumpp, H., Schubert, G.,
Hytham H. Gadalla: Methodology, Validation, Formal analysis, Chwalisz, K., 2003. Asoprisnil (J867): a selective progesterone receptor modulator
Writing - original draft. Fergany A. Mohammed: Conceptualization, for gynecological therapy. Steroids 68, 1019–1032.
Supervision. Ahmed M. El-Sayed: Conceptualization, Supervision. Dollery, C., 1999. Therapeutic Drugs, 2nd ed. Churchill Livingstone.
El-Gibaly, I., 2002. Oral delayed-release system based on Zn-pectinate gel (ZPG) micro-
Ghareb M. Soliman: Conceptualization, Supervision, Writing - review
particles as an alternative carrier to calcium pectinate beads for colonic drug de-
& editing. livery. Int. J. Pharm. 232, 199–211.
Gadalla, H.H., El-Gibaly, I., Soliman, G.M., Mohamed, F.A., El-Sayed, A.M., 2016a.
Amidated pectin/sodium carboxymethylcellulose microspheres as a new carrier for
Declaration of Competing Interest
colonic drug targeting: Development and optimization by factorial design.
Carbohydr. Polym. 153, 526–534.
The authors declare that they have no known competing financial Gadalla, H.H., Soliman, G.M., Mohammed, F.A., El-Sayed, A.M., 2016b. Development and
interests or personal relationships that could have appeared to influ- in vitro/in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for
the colonic delivery of progesterone. Drug Deliv. 23, 2541–2554.
ence the work reported in this paper. Jain, S.K., Jain, A., Gupta, Y., Ahirwar, M., 2007. Design and development of hydrogel
beads for targeted drug delivery to the colon. AAPS PharmSciTech 8, E34–E41.
Acknowledgement Jameela, S.R., Kumary, T.V., Lal, A.V., Jayakrishnan, A., 1998. Progesterone-loaded
chitosan microspheres: a long acting biodegradable controlled delivery system. J.
Control. Release 52, 17–24.
The authors would like to express their sincere gratitude for the late Jose, S., Dhanya, K., Cinu, T., Aleykutty, N., 2010. Multiparticulate system for colon
Dr. I. El-Gibaly (Department of Pharmaceutics, Faculty of Pharmacy, targeted delivery of ondansetron. Indian J. Pharm. Sci. 72, 58.
Kaur, A., Kaur, A., Kaur, P.V., Kaur, M., Murthy, R., 2014. Polymeric drug delivery ap-
Assiut University) for his insightful comments and contribution to this proaches for colon targeting: a review. Drug Deliv. Lett. 4, 38–48.
work. Also, Dr. Mahmoud Abdel-kareem (Department of Anatomy, Kim, J.Y., Kim, S., Papp, M., Park, K., Pinal, R., 2010. Hydrotropic solubilization of poorly
Embryology and Histology, Faculty of Veterinary Medicine, Assuit water-soluble drugs. J. Pharm. Sci. 99, 3953–3965.
Kowalonek, J., 2017. Studies of chitosan/pectin complexes exposed to UV radiation. Int.
University) is highly acknowledged for providing histological ex-
J. Biol. Macromol. 103, 515–524.
amination of tissue specimens. Maestrelli, F., Cirri, M., Corti, G., Mennini, N., Mura, P., 2008. Development of enteric-
coated calcium pectinate microspheres intended for colonic drug delivery. Eur. J.
Pharm. Biopharm. 69, 508–518.
Appendix A. Supplementary material
McPhail, M., 1934. The assay of progestin. J. Physiol. 83, 145–156.
Mennini, N., Furlanetto, S., Maestrelli, F., Pinzauti, S., Mura, P., 2008. Response surface
Supplementary data to this article can be found online at https:// methodology in the optimization of chitosan–Ca pectinate bead formulations. Eur. J.

8
H.H. Gadalla, et al. International Journal of Pharmaceutics 577 (2020) 119070

Pharm. Sci. 35, 318–325. study of PLGA and P (FASA) microspheres in vitro and in vivo. Biomaterials 23,
Mittal, G., Sahana, D., Bhardwaj, V., Kumar, M.R., 2007. Estradiol loaded PLGA nano- 4413–4423.
particles for oral administration: effect of polymer molecular weight and copolymer Schwarz, D.H., Engelke, A., Wenz, G., 2017. Solubilizing steroidal drugs by β-cyclodextrin
composition on release behavior in vitro and in vivo. J. Control. Release 119, 77–85. derivatives. Int. J. Pharm. 531, 559–567.
Nandi, I., Bateson, M., Bari, M., Joshi, H.N., 2003. Synergistic effect of PEG-400 and Seeli, D.S., Dhivya, S., Selvamurugan, N., Prabaharan, M., 2016. Guar gum succinate-
cyclodextrin to enhance solubility of progesterone. AAPS PharmSciTech 4, 1–5. sodium alginate beads as a pH-sensitive carrier for colon-specific drug delivery. Int. J.
Nappinnai, M., Sivaneswari, S., Swain, S., Behera, U.A., Beg, S., Sruti, J., Patro, C.N., Biol. Macromol. 91, 45–50.
Dinda, S., Rao, M., Mahajan, H.S., 2013. Formulation optimization and character- Sharma, S., Kaur, J., Sharma, G., Thakur, K.K., Chauhan, G.S., Chauhan, K., 2013.
ization of gastroretentive cefpodoxime proxetil mucoadhesive microspheres using 32 Preparation and characterization of pH-responsive guar gum microspheres. Int. J.
factorial design. J. Pharm. Res. 7, 304–309. Biol. Macromol. 62, 636–641.
Paharia, A., Yadav, A.K., Rai, G., Jain, S.K., Pancholi, S.S., Agrawal, G.P., 2007. Eudragit- Singh, S.K., Yadav, A.K., Prudhviraj, G., Gulati, M., Kaur, P., Vaidya, Y., 2015. A novel
coated pectin microspheres of 5-fluorouracil for colon targeting. AAPS PharmSciTech dissolution method for evaluation of polysaccharide based colon specific delivery
8, E87–E93. systems: A suitable alternative to animal sacrifice. Eur. J. Pharm. Sci. 73, 72–80.
Prasad, Y.V.R., Krishnaiah, Y.S.R., Satyanarayana, S., 1998. In vitro evaluation of guar Sinha, V.R., Kumria, R., 2003. Microbially triggered drug delivery to the colon. Eur. J.
gum as a carrier for colon-specific drug delivery. J. Control. Release 51, 281–287. Pharm. Sci. 18, 3–18.
Prezotti, F.G., Cury, B.S.F., Evangelista, R.C., 2014. Mucoadhesive beads of gellan gum/ Sriamornsak, P., Burton, M.A., Kennedy, R.A., 2006. Development of polysaccharide gel
pectin intended to controlled delivery of drugs. Carbohydr. Polym. 113, 286–295. coated pellets for oral administration: 1. Physico-mechanical properties. Int. J.
Ravi, V., Siddaramaiah, Pramod Kumar, T.M., 2008. Influence of natural polymer coating Pharm. 326, 80–88.
on novel colon targeting drug delivery system. J. Mater. Sci. Mater. Med. 19, Vinarov, Z., Dobreva, P., Tcholakova, S., 2018. Effect of surfactant molecular structure on
2131–2136. Progesterone solubilization. J. Drug Deliv. Sci. Technol. 43, 44–49.
Rodríguez, M., Vila-Jato, J.L., Torres, D., 1998. Design of a new multiparticulate system Wang, Q.-S., Wang, G.-F., Zhou, J., Gao, L.-N., Cui, Y.-L., 2016. Colon targeted oral drug
for potential site-specific and controlled drug delivery to the colonic region. J. delivery system based on alginate-chitosan microspheres loaded with icariin in the
Control. Release 55, 67–77. treatment of ulcerative colitis. Int. J. Pharm. 515, 176–185.
Ruan, X., Mueck, A.O., 2014. Systemic progesterone therapy—Oral, vaginal, injections Wang, X., Yu, D.-G., Li, X.-Y., Bligh, S.W.A., Williams, G.R., 2015. Electrospun medicated
and even transdermal? Maturitas 79, 248–255. shellac nanofibers for colon-targeted drug delivery. Int. J. Pharm. 490, 384–390.
Rubinstein, A., 2005. Colonic drug delivery. Drug Discov. Today Technol. 2, 33–37. Wiedmann, T.S., Liang, W., Kamel, L., 2002. Solubilization of drugs by physiological
Rubinstein, A., Radai, R., Ezra, M., Pathak, S., Rokem, J.S., 1993. In vitro evaluation of mixtures of bile salts. Pharm. Res. 19, 1203–1208.
calcium pectinate: a potential colon-specific drug delivery carrier. Pharm. Res. 10, Zarutskie, P.W., Phillips, J.A., 2009. A meta-analysis of the route of administration of
258–263. luteal phase support in assisted reproductive technology: vaginal versus in-
Sandor, M., Harris, J., Mathiowitz, E., 2002. A novel polyethylene depot device for the tramuscular progesterone. Fertil. Steril. 92, 163–169.